Osteoblast-specific deletion of NF-κB p65 enhances trabecular bone formation and BMP-2-induced osteogenesis in mice.

The nuclear factor κB (NF-κB) pathway plays critical roles in bone resorption and bone formation; however, the osteoblast-intrinsic function of NF-κB remains incompletely understood. In this study, we generated tamoxifen-inducible osteoblast-specific p65 (RelA)-deficient mice (p65ob-/-) using a Col1a1-CreERT2 system. Specific deletion of p65 in osteoblast-lineage cells was confirmed by immunohistochemical analysis. Primary analyses were performed using male mice, with selected findings validated in female mice. Osteoblast-specific deletion of p65 resulted in a significant increase in trabecular bone mass without affecting cortical bone, including approximately 20% higher total bone mineral density (total BMD), 25-30% higher trabecular bone mineral density (trabecular BMD), a 20-30% increase in bone volume fraction (BV/TV), a twofold increase in trabecular number (Tb.N), a 60-70% increase in trabecular thickness (Tb.Th), and a 50-60% reduction in trabecular separation (Tb.Sp). Histological analysis confirmed expansion of trabecular bone in the secondary spongiosa, and dynamic histomorphometry demonstrated increased bone formation rates, whereas osteoclast parameters remained unchanged. Similar skeletal phenotypes were observed in female mice, at later time points, and in the lumbar vertebrae. Furthermore, BMP-2-induced ectopic bone formation was enhanced in p65ob-/- mice. In primary osteoblast cultures, p65 deletion increased alkaline phosphatase activity under both basal osteogenic and BMP-2-stimulated conditions, whereas BMP-2-induced Smad1/5 phosphorylation remained unchanged. Collectively, these findings identify p65 as an intrinsic negative regulator of osteoblast-mediated bone formation and suggest that p65 suppresses osteoblast differentiation without altering BMP-2-induced Smad1/5 phosphorylation. These findings highlight p65-related pathways as potential targets for anabolic bone regeneration.